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A structure-function analysis of P2 integrase
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Elisabeth Haggård-Ljungquist)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Elisabeth Haggård-Ljungquist)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Elisabeth Haggård-Ljungquist)
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. (Elisabeth Haggård-Ljungquist)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Bacteriophage P2 integrase catalyzes site-specific recombination between the phage DNA and the host chromosome thereby promoting integration or excision of the phage genome. P2 integrase belongs to the large tyrosine family of integrases that shows little sequence identity besides some conserved boxes and patches in the catalytic domain. However, the overall structure of the tyrosine family of integrases seems to be similar. Phage integrases have the potential as tools for site-specific gene insertions into eukaryotic genomes provided that target sequences are available. To elucidate the possibility of evolving the P2 integrase to accept new targets, we have in this work initiated a structure-function analysis of the P2 integrase using two approaches based on a comparison of the predicted secondary structure of P2 integrase with that determined for the lambda integrase. First, we have made hybrids between P2 integrase and the related WΦ integrase that has a different host DNA target, to locate the region promoting specificity between the integrases. This, however, has not been possible, the N-terminal domains can be exchanged without losing biological activity and this will not affect the specificity. All other hybrids made were biological inactive. Next we have made an alanine scanning of the alpha helices believed to be involved in specific interactions with the target, and four amino acids have been identified as candidates for sequence-specific interactions with the core.

Keyword [en]
site-specific recombination, integrase, tyrosine recombinase, bacteriophage
National Category
Genetics Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-38927OAI: oai:DiVA.org:su-38927DiVA: diva2:317571
Note
Authors 1 and 2: Equal contribution to this workAvailable from: 2010-05-04 Created: 2010-05-04 Last updated: 2010-11-17Bibliographically approved
In thesis
1. Site-Specific Recombination: Integrases, Accessory Factors and DNA Targets of P2-like Coliphages
Open this publication in new window or tab >>Site-Specific Recombination: Integrases, Accessory Factors and DNA Targets of P2-like Coliphages
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The temperate coliphage P2 and its family members integrate their genomes into the host Escherichia coli chromosome by a site-specific recombination mechanism to form lysogeny. Integration takes place between the complex phage attP site and the simple bacterial attB site and is catalyzed by the phage encoded integrase (Int). Similar to the archetype λ Int, the P2-like phage integrases are heterobivalent tyrosine recombinases which possess the ability to simultaneously bind two different and distant types of DNA sequences within the attP region. To bridge the core and the flanking arm-binding sites in attP, the integrase requires the assistance of accessory factors that bend the DNA; the host encoded IHF and the phage encoded Cox protein. Cox acts as a directionality factor by being required for integration but is inhibitory for the excisive reaction.

The purpose of this doctoral thesis has been to gain a more detailed knowledge of the site-specific recombination systems of phages P2 and WΦ, which are close relatives but integrate into different host targets. The future aim is to develop these systems for targeted integration into the genome of higher eukaryotes.

The P2 Int and an N-terminal truncation of the integrase were shown to bind cooperatively together with IHF or Cox to the DNA targets, however the N-truncated protein lost its ability to bind to the arm sequence. WΦ Cox was shown to bind cooperatively with WΦ Int to attP whereas the opposite was evident for WΦ Cox and IHF. The 27 nucleotides that are identical between the core and attB of phage P2 were investigated for their importance in binding and recombination. The right part of the core was shown to be the primary Int binding site where one single base substitution was shown to abolish P2 Int binding and recombination. An alanine scanning of the two predicted alpha-helices in the presumed core-binding domain of P2 Int was carried out in order to identify amino acids involved in binding to the core. An in vivo excisive assay and an in vivo integrative assay were used resulting in the identification of four amino acids as candidates for core-binding. The fact that the recombination reaction shows directionality renders the site-specific recombination systems of the P2-like phages attractive to develop as tools for safe and efficient non-viral gene delivery in humans. The wild-type P2 integrase was shown to accept a human attB sequence and localizes to the nucleus in human cell lines.

The work presented in this thesis has increased our understanding of the site-specific recombination systems of the phages P2 and WΦ and provides a basis for further characterization and development for future use in a eukaryotic context.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2010. 51 p.
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-38914 (URN)978-91-7447-092-5 pp. 1-51 (ISBN)
Public defence
2010-06-04, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.Available from: 2010-05-11 Created: 2010-05-04 Last updated: 2010-05-12Bibliographically approved
2. Site-specific recombination of P2-like phages; possible tools for safe gene therapy: A focus on phage ΦD145
Open this publication in new window or tab >>Site-specific recombination of P2-like phages; possible tools for safe gene therapy: A focus on phage ΦD145
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

P2-like bacteriophages integrate their genome into the E. coli host cell by a site-specific recombination event upon lysogenization. The integrative recombination occurs between a specific sequence in the phage genome, attP, and a specific sequence in the host genome, attB, generating the host-phage junctions attL and attR. The integration is mediated by the phage enzyme integrase (Int) and the host factor IHF. The excisive recombination takes place between attL and attR, and is mediated by Int, IHF and phage encoded protein Cox. For safe integration of foreign genes into eukaryotic chromosome a recombinases is necessary which can perform the integration site-specifically. P2-like phage integrases have the potential to become tools for safe gene therapy. Their target is simple but specific, and once integration has occurred it is very stable in the absence of the Cox protein. The site-specific recombination mechanism has to be understood at the molecular level. Therefore, I have initiated the characterization of the site-specific recombination system of the P2-like phage ΦD145. In this work, Int and IHF are shown to bind to the different attachment sites cooperatively. One of two possible inverted repeats in attP is shown to be the Int core recognition site. The attP core of this phage has high identity with a site on human chromosome, denoted as ΨattB. In this study we have shown that in in vivo recombination ΦD145 Int can accept ΨattB in both bacteria and in eukaryotic cells. Also shown that Int consists of an intrinsic nuclear localization signal. A study also reveled that ΦD145 Int activity was affected by the Tyr-phosphorylation. Attempts have been made to change the specificity of the other P2-like phage P2 and WΦ integrases and also structural and functional analysis was done. A study on comparative analysis of Cox proteins and Cox binding sites gave us the basic information about the recombination mechanism.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2010. 50 p.
Keyword
bacteriophage, integrase, site-specific recombination
National Category
Natural Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-45940 (URN)978-91-7447-174-8 (ISBN)
Public defence
2010-12-17, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.Available from: 2010-11-25 Created: 2010-11-16 Last updated: 2010-11-17Bibliographically approved

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